Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion Wilson-Welder et al. Wilson-Welder et al. BMC Microbiology 2013, 13:280 http://www.biomedcentral.com/1471-2180/13/280 Wilson-Welder et al. BMC Microbiology 2013, 13:280 http://www.biomedcentral.com/1471-2180/13/280 RESEARCH ARTICLE Open Access Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion Jennifer H Wilson-Welder1,3*, Margaret K Elliott1, Richard L Zuerner1,3, Darrell O Bayles1, David P Alt1 and Thad B Stanton2 Abstract Background: Bovine papillomatous digital dermatitis (DD) is the leading cause of lameness in dairy cattle and represents a serious welfare and economic burden. Found primarily in high production dairy cattle worldwide, DD is characterized by the development of an often painful red, raw ulcerative or papillomatous lesion frequently located near the interdigital cleft and above the bulbs of the heel. While the exact etiology is unknown, several spirochete species have been isolated from lesion material. Four isolates of Treponema phagedenis-like spirochetes were isolated from dairy cows in Iowa. Given the distinct differences in host, environmental niche, and disease association, a closer analysis of phenotypic characteristics, growth characteristics, and genomic sequences of T. phagedenis, a human genitalia commensal, and the Iowa DD isolates was undertaken. Results: Phenotypically, these isolates range from 8.0 to 9.7 μm in length with 6–8 flagella on each end. These isolates, like T. phagedenis, are strictly anaerobic, require serum and volatile fatty acids for growth, and are capable of fermenting fructose, mannitol, pectin, mannose, ribose, maltose, and glucose. Major glucose fermentation products produced are formate, acetate, and butyrate. Further study was conducted with a single isolate, 4A, showing an optimal growth pH of 7.0 (range of 6–8.5) and an optimal growth temperature of 40°C (range of 29°C-43°C). Comparison of partial genomic contigs of isolate 4A and contigs of T. phagedenis F0421 revealed > 95% amino acid sequence identity with amino acid sequence of 4A. In silico DNA-DNA whole genome hybridization and BLAT analysis indicated a DDH estimate of >80% between isolate 4A and T. phagedenis F0421, and estimates of 52.5% or less when compared to the fully sequenced genomes of other treponeme species. Conclusion: Using both physiological, biochemical and genomic analysis, there is a lack of evidence for difference between T. phagedenis and isolate 4A. The description of Treponema phagedenis should be expanded from human genital skin commensal to include being an inhabitant within DD lesions in cattle. Keywords: Bovine digital dermatitis, Treponema, Spirochete, Bacterial growth, Genomic comparison Background to the farmer, especially when considering that a large Bovine papillomatous digital dermatitis (DD) is the pri- percentage of the herd may be affected [2,3]. Typical DD mary cause of lameness in dairy cattle and is a growing lesions are characterized by a rough, raw raised area concern to the beef industry [1]. Lameness attributed to most often occurring on the hind limb between the heel DD costs the producer $125-216/occurrence (treatment, bulb and dewclaw and may develop keratinaceous hair- lost productivity) representing a serious financial burden like projections. Lesions appear painful and are prone to bleeding when probed. Lesions generally do not heal * Correspondence: [email protected] spontaneously and may progress to severe lameness. 1Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Efficacious vaccines have so far been elusive [4,5]. Ames, Iowa 50010, USA Despite treatment and attempts at control, reoccurrence 3Mailing address: USDA, ARS, National Animal Disease Center, 1920 N. Dayton Ave, P.O. Box 70, Ames, IA 50010, USA of lesions both on the same hoof/cow and within the Full list of author information is available at the end of the article © 2013 Wilson-Welder et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wilson-Welder et al. BMC Microbiology 2013, 13:280 Page 2 of 9 http://www.biomedcentral.com/1471-2180/13/280 herd remains high [6]. Additionally, the welfare issue of maintaining food-producing animals in a healthy, pain- free state cannot be ignored [7]. Several Treponema species have been identified in tis- sue biopsies from DD lesions by in situ hybridization, immunohistochemistry and 16S rDNA sequence hom- ology [8-12]. Routinely, treponemes are found at the leading edge of lesions, deep within the tissue. Taking into account the spatial distribution of treponemes within the lesion and the robust immune response directed toward them [13-15], it is thought that these organisms may be key factors in DD lesion development. The goal of this study was to further characterize and compare laboratory growth characteristics, morphology, enzyme profiles, and draft genomic sequences of the T. phagedenis DD isolates, originally described by Trott et al. [14]. While these isolates share greater than 98% Figure 1 Negative stained electron photomicrograph of isolate 1A at 13000x magnification showing exposed flagella and 16S rDNA homology with T. phagedenis, with each insertion disks. Scale bar equal 500 nm. other, and with isolates from dairy herds in California [10], the United Kingdom [16], and Sweden [17], anti- genic variation and serological reactivity differ [13]. Pre- using the API ZYM system. Table 2 shows a comparison vious studies have focused on 16S rDNA analysis for of the enzyme activities of these isolates with T. phage- phylogenetic relatedness of Treponema isolates. Given denis, T. denticola, and other treponeme isolates. The T. differences in environmental niche and host species phagedenis-like DD isolates shared positive reaction for: between DD isolates and T. phagedenis type strains, we alkaline phosphatase, C4 esterase, C8 esterase lipase, acid sought to compare the physical appearance, growth rate, phosphatase, naptholphosphohydrolase, β-galactosidase, biochemical substrates, and draft genomes. Results of and N-acetyl-β-glucosaminidase. These results matched these studies and genome-wide comparisons indicate the T. phagedenis biovar Kazan reactivity profile, except that T. phagedenis-like isolates from DD lesions of cattle that Kazan additionally tested positive for leucine aryla- are nearly identical to T. phagedenis, suggesting an ex- midase activity. The two biovars of T. phagedenis (Kazan pansion of environmental niches occupied by this bac- and Reiter) differed in 6 of the API ZYM tests from each terium. We propose the description of T. phagedenis be other and are known to differ in enzymatic activity [18]. expanded to include both human commensal and puta- In contrast, T. denticola differed in six different enzym- tive bovine pathogen. atic reactions from the Iowa DD isolates. Assay variabil- ity is clearly demonstrated as in this study T. denticola Results showed positive reactivity for C8 esterase lipase, acid Morphology phosphatase, naptholphosphohydrolase, α-galactosidase, Morphological characteristics were determined by phase and α-glucosidase where the same strain published else- contrast, dark field, and electron microscopy. Cells were where was negative for these 5 enzymes but positive for grown in OTI and visualized directly from log-phase chymotrypsin [19]. Although assay subjectivity and vari- culture by phase contrast and dark field microscopy. ations in methodology make cross-laboratory compari- Cells exhibited typical helical morphology with a slight sons difficult, the API-ZYM profile for Iowa DD isolates flattening of the pitch at one or both ends of the cell. closely match the published profile for T. phagedenis Both rotating and translational motility was observed and T. brennaborense as well as several other T. under dark field microscopy. As determined by electron microscopy, cell dimensions of isolates 1A, 3A, 4A and Table 1 Size and flagella number for Iowa isolates as 5B varied from 8 to 9.7 μm in length and 0.3 to 0.35 μm determined by electron microscopy in width, with 7 to 9 flagella attached on terminal ends Isolate Isolate Isolate Isolate T. phagedenis with 7-14-7, 8-16-8 or 9-18-9 arrangements (Figure 1, 1A 3A 4A 5B Kazan Table 1). Length (μm) ± 8.0 ± 0.8 8.7 ± 1.3 9.7 ± 2.6 9.4 ± 0.9 10.4 ± 0.9 StdDev API ZYM profile Flagella number 7.3 ± 1.2 7.3 ± 0.5 8.7 ± 0.9 6.6 ± 0.9 6.9 ± 1.2 The enzyme activity profiles of the four Iowa isolates (single end) ± StdDev and the reference treponeme species were determined Wilson-Welder et al. BMC Microbiology 2013, 13:280 Page 3 of 9 http://www.biomedcentral.com/1471-2180/13/280 Table 2 Comparison of API-ZYM substrate reactivity profiles of Iowa isolates against other DD isolates and known Treponema strains 1234 5 678910111213141516171819 Iowa Isolates 1A, 3A, 4A & 5B* +++------++-+- - -+- - T. phagedenis Kazan* + + + - + - - - - + + - + - - - + - - T. phagedenis Reiter§ - - - - - - - - - +